Project Summary Myelination of the central nervous system (CNS) by oligodendrocytes (OLs) is essential for the development and function of the CNS. Genetic studies have shown that Olig1/2, basic helix-loop-helix transcription factors, are master regulators of OL lineage cells, playing an indispensable role in the development and regeneration of CNS myelin. A fundamental yet unresolved issue surrounding Olig1/2 is who regulates their expression. Apparently, for proper OL development, they have to be expressed at the right level at the right moment in OL lineage cells. What regulatory mechanism ensures it? The first critical step toward this grand challenge is to identify OL enhancers that govern their expression. Historically, finding enhancers for a gene has been difficult. To make a breakthrough for this fundamental issue, we have developed a novel method that takes advantage of recent advances in our understanding of genome organization and enhancers. We have demonstrated its power by applying it to Myrf, uncovering two OL enhancers, including one novel one, that jointly regulate Myrf expression for timely differentiation of OLs. This grant will capitalize on this new powerful method in order to identify OL enhancers that govern the expression of Olig2 (Aim I) and Olig1 (Aim II). .